G-Protein Coupled Receptors
Many medically significant biological processes are mediated by signal transduction pathways that involve G-proteins (Lefkowitz, Nature 351, 353-354, 1991). The family of G-protein coupled receptors (GPCR) includes receptors for hormones, neurotransmitters, growth factors, and viruses. Specific examples of GPCRs include receptors for such diverse agents as dopamine, calcitonin, adrenergic hormones, endothelin, cAMP, adenosine, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorants, cytomegalovirus, G-proteins themselves, effector proteins such as phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins such as protein kinase A and protein kinase C.
GPCRs possess seven conserved membrane-spanning domains connecting at least eight divergent hydrophilic loops. GPCRs (also known as 7TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. Most GPCRs have single conserved cysteine residues in each of the first two extracellular loops, which form disulfide bonds that are believed to stabilize functional protein structure. The seven transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.
Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some GPCRs. Most GPCRs contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several GPCRs, such as the •-adrenergic receptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
For some receptors, the ligand binding sites of GPCRs are believed to comprise hydrophilic sockets formed by several GPCR transmembrane domains. The hydrophilic sockets are surrounded by hydrophobic residues of the GPCRs. The hydrophilic side of each GPCR transmembrane helix is postulated to face inward and form a polar ligand binding site. TM3 has been implicated in several GPCRs as having a ligand binding site, such as the TM3 aspartate residue. TM5 serines, a TM6 asparagine, and TM6 or TM7 phenylalanines or tyrosines also are implicated in ligand binding.
GPCRs are coupled inside the cell by heterotrimeric G-proteins to various intracellular enzymes, ion channels, and transporters (see Johnson et al., Endoc. Rev. 10, 317-331, 1989). Different G-protein alpha-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of GPCRs is an important mechanism for the regulation of some GPCRs. For example, in one form of signal transduction, the effect of hormone binding is the activation inside the cell of the enzyme, adenylate cyclase. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP. GTP also influences hormone binding. A G-protein connects the hormone receptor to adenylate cyclase. G-protein exchanges GTP for bound GDP when activated by a hormone receptor. The GTP-carrying form then binds to activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
Over the past 15 years, nearly 350 therapeutic agents targeting GPCRs receptors have been successfully introduced onto the market. This indicates that these receptors have an established, proven history as therapeutic targets. Clearly, there is an on-going need for identification and characterization of further GPCRs which can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, infections such as bacterial, fungal, protozoan, and viral infections, particularly those caused by HIV viruses, pain, cancers, anorexia, bulimia, asthma, Parkinson's diseases, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, several mental retardation, and dyskinesias, such as Huntington's disease and Tourett's syndrome.
Neuropeptide Y
Neuropeptide Y (NPY) is a 36-residue, amidated polypeptide. It is anatomically co-distributed and co-released with norepinephrine in and from sympathetic postganglionic neurons (11, 2, 3, 4, 5, and 6). Stimulation of the sympathetic nervous system under physiological circumstances such as exercise (7, 8) or exposure to the cold (9, 10) promotes an elevation of both norepinephrine and NPY.
NPY is believed to act in the regulation of appetite control (11, 12) and vascular smooth muscle tone (13, 14), as well as regulation of blood pressure (6, 15, 16, 17). NPY also decreases cardiac contractility (18, 19, 20, 21, 22). Congestive heart failure and cardiogenic shock are associated with probable releases of NPY into the blood (23, 24, 25). Regulation of NPY levels may be beneficial to these disease states (26).
At the cellular level, NPY binds to a G-protein coupled receptor (GPCR) (27, 28, 29, 30). NPY is involved in regulating eating behavior and is an extremely potent orixigenic agent (11, 12, 31). When administered intracerebroventricularly or injected into the hypothalamic paraventricular nucleus (PVN) it elicits eating in satiated rats (32, 33, 34) and intraventricular injection of antisera to NPY decreases eating (11, 31). It has been shown to stimulate appetite in a variety of species and at different stages of development (12). Other effects on energy metabolism include decreased thermogenesis, body temperature, and uncoupling protein, and increased white fat storage and lipoprotein lipase activity (9, 35, 36, 37, 38, 39). NPY levels in the PVN increase upon fasting (40, 41, 42, 43, 44), before a scheduled meal (31, 36, 40), and in both streptozotocin-induced and spontaneous diabetes (36, 45, 46, 47, 48, 49). Also, NPY levels are increased in genetically obese and hyperphagic Zucker rats (36, 50, 51). Thus, a specific centrally acting antagonist for the appropriate NPY receptor subtype may be therapeutically useful for treating obesity and diabetes. Other disorders which can be targeted therapeutically include anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's, and Parkinson's diseases (26, 52).
Neuropeptide Y Receptors
At least four receptor subtypes of the NPY family have been proposed based on pharmacological and physiological properties. The Y1 receptor is stimulated by NPY or PYY (peptide YY) and appears to be the major vascular receptor (16, 53, 54, 55). The Y2 receptor is stimulated by C-terminal fragments of NPY or PYY and is abundantly expressed both centrally and peripherally (55, 56, 57, 58). A third receptor (Y3) is exclusively responsive to NPY and is likely present in adrenal medulla, heart, and brainstem (27, 59). In addition, other subtypes of this receptor family are known to exist, based on pharmacological and physiological characterization (60, 61, 62, 63). The feeding behavior is stimulated potently by NPY, NPY2-36, and the Y1 agonist Leu31, Pro34 NPY, but is not stimulated by the Y2 agonist NPY13-36 (11, 64, 65, 66). This pharmacology is not characteristic of the defined Y1, Y2, or Y3 receptors and can thus be attributed to a unique receptor, termed “atypical Y1” (11, 65, 66), which is responsible for evoking the feeding response. In addition, data indicate the existence of additional members of this receptor family, including one subtype specific for peptide PP (62, 63), one with affinity for short C-terminal fragments of NPY which induce hypotension when administered systemically (15, 17, 30, 67, 68), and one associated with binding of NPY and PYY to brain sigma and phencyclidine binding sites (61).
The Y1 receptor has been cloned and shown to be a G-protein coupled receptor (53, 69, 70). Recently, the Y2 receptor has been cloned (71, 72). In addition, a peptide PP-preferring receptor, termed PPI (73) or Y4 (74), has been cloned. Other NPY receptors also have been recently identified and cloned. See U.S. Pat. Nos. 5,939,263 and 5,965,392.
Because of the diverse biological effects of neuropeptide Y receptors, there is a need in the art to identify additional members of the neuropeptide Y receptor family whose activity can be regulated to provide therapeutic effects.